Our surroundings are extremely rich in spectral information, which confers a valuable chromatic dimension to visual perception. In order to have the capacity for color vision, an organism must be able to perform the necessary computations to compare light of different spectral compositions. Wavelength comparison takes place in color opponent neurons, which respond with opposite polarity to wavelengths in different parts of the spectrum. Despite advances in our understanding of color opponency in the brain, how color opponent signals are transformed to give rise to the hue specificity observed in higher cortical regions remains completely unexplained. Furthermore, how wavelength information is integrated across the visual field to provide a spatial dimension to color vision is a poorly understood phenomenon. This project aims to examine color pathways in the genetically tractable organism Drosophila melanogaster, as these circuits have only just begun to be described. Drosophila provide an arsenal of genetic tools to manipulate neuronal activity and a simple brain that makes these circuits tractable. Fruit flies have the hardware for wavelength comparison, with wavelength-specific photoreceptors (called R7s and R8s) expressing rhodopsins sensitive to UV, green, and blue light. There is mounting evidence that color opponency is indeed present in the brain of the fruit fly, arising in the axons of R7/R8. Aim 1 will determine how signals are combined at the level of photoreceptors to give rise to opponency by using two-photon calcium imaging of R7/R8 axons in a variety of genetic backgrounds, including mutants, pairwise rescues, and lines with cell-specific silencing. Aim 2 will elucidate the spatial nature of opponency in Drosophila photoreceptors, taking advantage of the fact that spatially patterned stimuli will reveal potential center-surround mechanisms when paired with functional imaging of R7/R8 axons. Finally, Aim 3 will explore the encoding of both spectral and spatial information in downstream brain areas poised to both receive signals from photoreceptors, and to further transmit these signals to central brain regions. There is evidence that this information eventually informs tasks such as object recognition and spatial orientation. Determining how circuit mechanisms for spatio-chromatic processing emerge and convey information to higher brain areas in Drosophila will provide insight into the workings of vertebrate color pathways, as both systems employ similar mechanisms to effectively process visual information.